We have found that nicking-closing enzymes isolated from eukaryotic cells can mediate the assembly of histones and DNA to form chromatin under physiological conditions in vitro. We propose to analyze the molecular mechanism of this chromatin assembly in order to determine the functional role of the nicking-closing enzyme in the process. We will also examine the mechanism of chromatin assembly that is catalyzed by a crude extract of preblastoderm embryos of Drosophila melanogaster. This extract contains a pool of free maternal histones which can interact with DNA to from chromatin, again at physiological conditions of salt and temperature. Moreover, this extract is capable of assembling 100 fold higher amounts of chromatin if exogenous histones are supplied. By fractionation and purification of the chromatin assembly factors we will determine the mechanism of this assembly process and in particular ask whether the nicking-closing activity which is present in these extracts plays a role in assembly. In addition to the classical nicking-closing enzyme found in all eukaryotic sources, these preblastoderm embryos contain very high levels of an ATP-dependent DNA topoisomerase. This enzyme, together with another DNA binding protein is capable of converting circular DNAs into topologically linked catenated dimers. This reaction does not require homology and catenated dimers and higher oligomers of nonhomologous DNAs can be formed. We will investigate the enzymatic mechanism of the formation of catenated dimers and the role played by ATP. In the absence of the DNA-binding protein, the ATP-dependent topoisomerase alone is capable of resolving catenated structures back into their monomer form in a reaction that also requires ATP. Both of these reactions, the formation and the resolution of catenated structures, could be very useful mechanisms for the folding and segregation of eukaryotic chromosomes. We hope to determine the molecular mechanism whereby this ATP-dependent DNA topoisomerase allows one DNA helix to pass through another.